U.S. Pat. No. 3,092,463 discloses the use of strips for analyzing some blood constituents. These strips are made of filter paper impregnated with different reagents such as encapsulated hydroperoxides, an indicator (o-tolidine) and a buffer (citrate). Under normal storage conditions, no reaction occurs between the reagents and no discoloration of the indicator is observed but when the strips are dipped into a solution containing a substance to be analyzed, e.g. the prosthetic groups of blood, the capsules break hydrolytically with consecutive liberation of the hydroperoxide and a color reaction with the indicator is allowed to develop. However, these strips have the disadvantages that the filter paper (bibulous material) which supports the reagents often contains impurities which may interfere with the color development and, further, its sampling capacity for the solution to be analyzed is not well constant from test to test because of unavoidable differences in the fibrous material used for making the strips; thus the method is only qualitative. Another disadvantage is that the encapsulation is performed by means of a colloid substance such as gelatin, gum arabic or carboxy-vinyl polymers that produces very fragile thin walled capsules of questionable mechanical and storage stability. Another disadvantage is that the color will develop within the bibulous material itself which is often opaque and not homogeneous, thus introducing further sources of errors in the color evaluation.
U.S. Pat. No. 3,926,732 discloses the use of test strips for the color determination of catalase in milk. This method is based on the catalase dependent inhibition of the color reaction involved upon oxidation of a leuko-dye (o-tolidene) by hydrogen peroxide in the presence of peroxidase. In one embodiment of said reference, the strips comprise an inert solid absorption and diffusion medium in which are embedded physically separated individual microcapsules. The microcapsules are of the "Semi-permeable aqueous" type (see Can. J. Physiol. Pharmacol. 44 (1966), 115) and the medium is made of cellulose fibers. When the strip is dipped into the solution to be analyzed, the hydrogen peroxide generated by the reagents of a first kind of microcapsules will migrate through the medium whereby it will be partially inhibited in proportion to the amount of catalase to be analyzed and, finally, it will react with the dye having diffused from microcapsules of a second kind. The color will then be released within the diffusion medium with consecutive disadvantages as mentioned heretofore. Also in this method the sampling capacity of the strip per unit area is very difficult to keep constant and the method is not accurate. Another disadvantage of the strips involving layers of fibrous absorbing media is the face that such media have a largely opened structure which does not adequately filter out unwanted colored species which may be present in the liquid to be analyzed and which may interfere with the desired color reaction. This has been partly remedied by using strips with a low porosity media layer in which the reagents are dispersed or dissolved. Such kinds of strips are disclosed for instance in U.S. Pat. No. 3,630,957, but they have the drawback that they possess no instant sampling capacity for the liquid to be analyzed. Indeed, when such strips are immersed in said liquid, the latter will slowly penetrate into the pores of the medium (in the range of 0.1-1 .mu.m) and the substances to be analyzed will diffuse toward the reagents leaving out the possibly interfering materials. However, this process will take several minutes or more and it is not possible to exactly say at which time the process is really effective and when the color is sufficiently developed. Therefore, the test is slow and qualitative only. Some of the above drawbacks can be obviated by depositing a drop of the liquid to be analyzed on the strip. However, this is also a slow process and there is no way to tell that the drop will always be absorbed by a known given area of the strip; therefore the test is also qualitative only.
The above disadvantages have been partly cured by using the strips disclosed in French Pat. No. 2,303,290. These strips comprise as a diffusing medium a synthetic polymer obtained by the phase inversion precipitation technique. According to this technique, a solution of a polymer is prepared in a mixture of two solvents, one of which being a poorer solvent for this polymer and less volatile than the other solvent. When the solution is allowed to dry, there becomes a moment when the good solvent has sufficiently evaporated for causing the polymer to slowly precipitate which results, after complete drying, in an opened porous gelled bibulous structure resembling cellulose fibers and having favorable sampling properties for the liquid to be analyzed. The reagents necessary to develop the analytical color reactions of the strips can be either incorporated by impregnation or by dissolving into the original polymer solution. This is advantageous because, in case of mutually incompatible reagents, one of which can be put within the body of the polymer itself whereas the other can be imbibed in the opened porous structure whereby contact between said reagents in the dry state under storage is minimized. These strips have many advantages, however the intimate structure and the porosity of the matrix is strongly dependent on the preparative conditions and reproducibility of the specifications is difficult to maintain from batch to batch. Also, because of its rather fibrous nature, this material can introduce differential diffusion effects on the solution to be analyzed which can be a source of inconsistency in color development. Further, the color develops within the full body of the absorptive medium which, because of light diffraction problems related to the structure thereof, may limit the sensitivity in some cases.
U.S. Pat. No. 3,993,451 discloses test strips an embodiment of which comprises a strip of hydrophilic paper coated, on one side, with a uniform layer of a homogeneous mixture of two kind of particles. These particles are formed of agglomerates of hydrophilic absorbent materials such as powdered cellulose, alumina, silicagel, etc . . . impregnated by the reagents solutions and dried thereafter. One kind of the particles contain a first reagent and the second kind of particles contain a second reagent, said two reagents being not compatible and being thus kept apart during storage. When dipped into the solution to be analyzed, a portion of said solution is sampled by the absorbent materials and the reagents are allowed to contact the substance to be tested with consecutive development of the color reaction. This color test is therefore very fast but the sampling action is rather erratic because of the powdery nature of the absorbent material and the uncontrolled size of the agglomerates containing the reagents.
The present invention has for object to correct as much as possible the above discussed deficiencies. Briefly summarized an ideal test strip would fullfil the following needs and properties:
(a) Accurately sample a given amount of solution to be analyzed per unit area of the strip.
(b) Achieve such sampling instantaneously in the course of one single move: dip and withdraw.
(c) Allow intimate contact between the substance to be analyzed and the reagents and filter out undesirable component of the solution which may interfere with the desired analytical reactions.
(d) Allow rapid and reproducible color development with good sensitivity and good measurability (visual or spectrophotometer).
(e) Allow good separation under storage of reagents when mutually incompatibles.
(f) Accomodate reagents of different compatibilities, e.g. water-soluble reagents and liposoluble reagents.
(g) Avoid as much as possible so called "inert" media which may mask or interfere with the color reaction.
(h) Offer simple and economic fabrication routes.
(i) Be strong enough mechanically to withstand accidental abuse (rupture by abrasion).
(j) Have a long shelf-life, i.e. have reagents well shielded and preserved from evaporation or decomposition.